Activation of naïve T cells is thought to proceed by a two-signal mechanism. Upon encountering an antigen presenting cell (APC), the T cell receptor (TCR) interacts with peptide in the context of major histocompatibility complex (MHC) molecules and thus delivers the first activation signal to the T cells. This initial signal is insufficient to lead to T cell activation and a second signal from co-stimulatory receptors is an absolute requirement. One of the most important and best described co-stimulatory receptor is CD28 which interacts with CD80 (B7.1) and CD86 (B7.2) on the surface of macrophages, dendritic cells as well as B and activated T lymphocytes.
The CD86 gene encodes a type I membrane protein (Swiss-Prot Acc-No P33681). Alternative splicing results in two transcript variants of the CD86 gene encoding different isoforms. Additional transcript variants have been described, but their full-length sequences have not been determined.
The related protein CD80 (Swiss-Prot Acc-No P42081) has a secondary structure similar to CD86. CD80 shares 26% and 46% of identical and similar amino acid residues with CD86, respectively. CD80 is expressed only at low levels in resting APCs but can be up-regulated following activation. CD80 recognizes the same receptors on T cells, CD28 and CD152 (CTLA-4), but binds the latter with approximately 2 to 4 fold higher affinity than CD86 does.
No shared linear peptide epitope had been identified that is responsible for binding to CD28 and/or CTLA-4 (Ellis et al., J Immunol., 156, 2700-2709) but conserved residues in the secondary structures (IgV sheets of CD80 and CD86) had been found in interaction with CTLA-4 (Swartz et al., Nature, 410, 604-608).
Signal transduction from CD28 leads to T cell activation and the upregulation of the CTLA-4 co-inhibitory receptor. CTLA-4 is a member of the immunoglobulin superfamily. It binds to CD80 and CD86 with increased affinity and avidity compared with CD28 and effectively downregulates activation signals.
Various theories have been postulated on the relative roles of CD80 and CD86 in binding CTLA-4. Slavik et al. (Immunol. Res. 19(1):1-24 1999) reviewed the signalling and function of the CD28/CTLA-4 and CD80/CD86 families. Sansom (Immunology 101:169-177 2000) summarised some studies where differences between CD80 and CD86 were investigated.
Odobasic et al. (Immunology 124:503-513 2008) investigated the roles of CD80 and CD86 in effector T cell responses. This study investigated the effects of anti-CD80 and anti-CD86 monoclonal antibodies in an antigen-induced mouse model of arthritis. It was reported that blockade of both CD80 and CD86 caused a trend towards reduced disease severity compared to control antibody-treated mice. Based on the results of treatment with the individual antibodies, the authors concluded that CD80 exacerbates arthritis by downregulating systemic IL-4 and increasing T cell accumulation in joints, while CD86 enhances disease severity by upregulating IL-17 and increasing the accumulation of effector T cells in joints without affecting Th1 or Th2 development. However, the study reports that no further additive reduction in arthritis severity was observed when both CD80 and CD86 were blocked, suggesting that inhibition of either costimulatory molecule was adequate to obtain maximal disease amelioration. This model was based on a recall response to antigen (BSA in this study) directly injected in the joint space.
Another study used a murine collagen-induced arthritis model, involving breaking tolerance to an endogenous antigen (collagen). In this study, blockade of both CD80 and CD86 was reported to be required for maximal benefit (Webb et al. Eur J. Immunol 26(10):2320-2328 1996).
A recombinant fusion protein comprising the extracellular domain of CTLA-4 linked to a modified IgG1 Fc domain (“CTLA-4-Ig”) has been shown to bind CD80 and CD86 in vivo and effectively suppress CD28-mediated T cell activation (Kliwinski et al., J Autoimmun. 2005; 25(3):165-71).
CTLA-4 fusion proteins have been developed as therapeutic agents for rheumatoid arthritis (RA). RA is a progressive degenerative disease leading to cartilage and bone destruction. There is evidence that many arms of the immune system are involved in the inflammatory process leading to fibroblast-like synoviocytes and osteoclast-mediated joint damage and cartilage and bone destruction. Multiple studies have shown increased T cell activation in the synovium and up to 50% of the cells infiltrating the inflamed pannus are T lympho. Furthermore, T cells in the synovium of RA patients exhibit an activated effector phenotype displaying increased expression of activation associated markers such as CD44, CD69, CD45RO, VLA-1 and CD27.
Activated T cells have been shown to play a key role in establishing and maintaining the pathological inflammatory response found in the RA synovium. Activated T cells are an important source of proinflammatory cytokines, such IFNγ, IL-17 and TNFα. These factors are potent activators of fibroblast-like synoviocytes (FLS) and macrophage-like synoviocytes (MLS) leading to the secretion of matrix metalloproteinases (MMP) which are mediators of cartilage destruction as well as the secretion of inflammatory mediators such as IL-6, IL-1 and TNFα. Activated CD4+ cells may also provide cognate help to B lymphocytes leading to the production of antibodies, such as rheumatoid factor (RF), that further contribute to disease progression.
Abatacept (ORENCIA®) is a CTLA-4 Ig fusion protein containing the extracellular domain of CTLA-4 fused to the Fc of IgG1. The resulting soluble protein is a dimer with a molecular weight of approximately 92 kDa. It has been shown to have beneficial effects in treating RA patients in the clinic, demonstrating that inhibition of the co-stimulation pathway involving CD80 and CD86 is a viable therapeutic approach for RA. RA therapy with Abatacept is administered either as an intravenous monthly or a weekly subcutaneous injection.
Abatacept contains in its CDR3-like loop the amino acid hexapeptide motif MYPPPY, which is shared between CD28 and CTLA-4 and is reported to be necessary for binding to the B7 ligands. Mutation of the first tyrosine (Y) in this motif to alanine (A) abolishes binding to CD80, but also results in reduced binding to CD86, whereas a phenylalanine (F) substitution allows for retention of the full affinity for CD80 with a total loss of CD86 binding (Harris et al., J. Exp. Med. (1997) 185:177-182). Other residues in the CDR3-like and CDR1-like regions are also important for the interaction of Abatacept with its ligands. Thus, a mutant molecule with glutamic acid (E) instead of leucine (L) at position 104 and tyrosine (Y) instead of alanine (A) at position 29 exhibits approximately 2-fold greater binding avidity for CD80 (B7-1) and approximately 4-fold greater binding avidity for CD86 (B7-2) than abatacept. This compound LEA-29Y (Belatacept, NULOJIX®; a selective T-cell (lymphocyte) costimulation blocker, that binds to CD80 and CD86 on antigen-presenting cells thereby blocking CD28 mediated costimulation of T lymphocytes) is reported to have similar affinity for binding CD80 as for binding CD86 (3.66 nM and 3.21 nM respectively). Belatacept has been developed as an immunosuppressant for transplantation (Larsen et al., Am. J. Transplantation (2005) 5:443-453; Gupta & Womer Drug Des Develop Ther 4:375-382 2010) and was recently approved for prophylaxis of organ rejection in adult patients receiving a kidney transplant. Abatacept itself showed limited efficacy against transplant rejection, a finding that has been attributed to its lower inhibition of CD86-dependent as opposed to CD80-dependent costimulation (Gupta & Womer, supra).
Formulations of Abatacept and Belatacept for subcutaneous administration are described in WO2007/07654.
Selections for improved affinity and stability have previously been performed using ribosome display to isolate improved variants of CTLA-4. Both error-prone PCR mutagenesis, to mutate the entire gene sequence, and directed mutagenesis, to target mutations to key regions, have been successful for protein evolution. For example, WO2008/047150 reported protein variants of CTLA-4 showing increased activity and increased stability compared with wild type.
Maxygen, Inc. reported a CTLA-4-Ig therapeutic molecule, designated ASP2408, being developed by Perseid Therapeutics LLC in collaboration with Astellas Pharma Inc for the treatment of RA. The CTLA-4-Ig was reported to show improved potency compared with ORENCIA® (Abatacept) a CTLA-4 Ig fusion protein containing the extracellular domain of CTLA-4 fused to the Fc of IgG1 (WO2009/058564).
U.S. Pat. No. 6,750,334 (Repligen Corporation) described CTLA-4-Cγ4, a soluble fusion protein comprising CTLA-4 fused to a portion of an immunoglobulin. The immunoglobulin constant region, comprising a hinge region and CH2 and CH3 domains, is modified by substitution, addition or deletion of at least one amino acid residue, to reduce complement activation or Fc receptor interaction.
Xencor, Inc. recently described a CTLA4-Ig molecule comprising a variant CTLA-4 portion and an immunoglobulin Fc region (WO2011/103584). A number of amino acid substitutions in the amino acid sequence of the CTLA-4 portion were described, for generating CTLA4-Ig variants with greater T-cell inhibitory activity. WO2011/103584 also describes Fc modifications, for example for improving binding to FcγRs, enhancing Fc-mediated effector functions and/or extending in vivo half life of the CTLA4-Ig.